Abstract: Hapalogenys nitens is a kind of delicious marine fish, mainly distributes in China, Japan and Korea, which has become an important culture fish in China. In recent years, as economic activity is becoming more frequent, fewer and fewer natural nitens could be captured in Guangdong, Fujian, Zhejiang and other provinces along the coast. It is important to analyze the population genetic structure of Hapalogenys nitens. For evaluating the genetic diversity of Hapalogenys nitens, 50 individuals of Hapalogenys nitens were collected from Guangdong province. All of the 50 individuals were come from two groups which were wild and cultured groups. Half of them was the wild population, and the other half was the cultured population. Study on genetic diversity evaluation of fish is mainly concentrated on gene mutation in nucleus. But in recent years, the role of mitochondrial DNA (mtDNA) mutation in genetic diversity research has been given more attention. Mitochondrial DNA (mtDNA) is known for high mutation rates caused by lack of protective histones, inefficient DNA repair systems, and continuous exposure to mutagenic effects of oxygen radicals. So the analysis of mitochondrial DNA has been a potent tool in our understanding of Hapalogenys nitens genetic diversity between wild and cultured populations. The D-loop occurs in the main non-coding area of the mitochondrial DNA molecule, a segment called the control region or D-loop region. In this study, we chose the mtDNA D-Loop region for genetic diversity evaluation and examined the frequency of mutations in the mtDNA D-Loop region in 50 individuals. The mitochondrial DNA of them were extracted, and the mtDNA D-Loop region of 50 individuals were sequenced for the comparative analysis. Sequences length of the D-loop area was from 788 bp to 790 bp in 50 Hapalogenys nitens individuals by PCR amplification and electrophoresis. The sequences were analyzed by using the MEGA (version 4.0) and DnaSP (version 4.0) software. The result showed that the average content of T, C, A and G was 29.9%, 21.5%, 35.2% and 13.5% in the D-loop area of mtDNA, respectively. In these 50 haplotype samples, there were 91 polymorphic sites in this area. And the average genetic distance of haplotypes, the haplotypic diversity and the nucleotide diversity were 0.024, 0.958 and 0.02277 respectively. It implied that the D-loop area of mtDNA could be used as the molecular markers on evaluating the genetic diversity in the wild and cultured Hapalogenys nitens population, and the genetic diversity of the Hapalogenys nitens population was at a medium level in Nan'ao District, Shenzhen City, Guangdong province. These findings suggested that the genetic diversity of the wild Hapalogenys nitens was not optimistic; especially, the research which focused on the genetic evaluation, the resource maintenance and survey of the Hapalogenys nitens need to be further studying in the future.